The present invention is in the fields of molecular and cellular biology. The invention is generally related to reverse transcriptase enzymes and methods for the reverse transcription of nucleic acid molecules, especially messenger RNA molecules. Specifically, the invention relates to compositions comprising mixtures of reverse transcriptase enzymes, and to methods of producing, amplifying or sequencing nucleic acid molecules (particularly cDNA molecules) using these reverse transcriptase enzymes or compositions. The invention also relates to nucleic acid molecules produced by these methods and to the use of such nucleic acid molecules to produce desired polypeptides. The invention also concerns kits comprising such compositions.
cDNA and cDNA Libraries
In examining the structure and physiology of an organism, tissue or cell, it is often desirable to determine its genetic content. The genetic framework of an organism is encoded in the double-stranded sequence of nucleotide bases in the deoxyribonucleic acid (DNA) which is contained in the somatic and germ cells of the organism. The genetic content of a particular segment of DNA, or gene, is only manifested upon production of the protein which the gene encodes. In order to produce a protein, a complementary copy of one strand of the DNA double helix (the xe2x80x9ccodingxe2x80x9d strand) is produced by polymerase enzymes, resulting in a specific sequence of ribonucleic acid (RNA). This particular type of RNA, since it contains the genetic message from the DNA for production of a protein, is called messenger RNA (mRNA).
Within a given cell, tissue or organism, there exist myriad mRNA species, each encoding a separate and specific protein. This fact provides a powerful tool to investigators interested in studying genetic expression in a tissue or cellxe2x80x94 mRNA molecules may be isolated and further manipulated by various molecular biological techniques, thereby allowing the elucidation of the full functional genetic content of a cell, tissue or organism.
One common approach to the study of gene expression is the production of complementary DNA (cDNA) clones. In this technique, the mRNA molecules from an organism are isolated from an extract of the cells or tissues of the organism. This isolation often employs solid chromatography matrices, such as cellulose or agarose, to which oligomers of thymidine (T) have been complexed. Since the 3xe2x80x2 termini on most eukaryotic mRNA molecules contain a string of adenosine (A) bases, and since A binds to T, the mRNA molecules can be rapidly purified from other molecules and substances in the tissue or cell extract. From these purified mRNA molecules, cDNA copies may be made using the enzyme reverse transcriptase (RT), which results in the production of single-stranded cDNA molecules. The single-stranded cDNAs may then be converted into a complete double-stranded DNA copy (ie., a double-stranded cDNA) of the original mRNA (and thus of the original double-stranded DNA sequence, encoding this mRNA, contained in the genome of the organism) by the action of a DNA polymerase. The protein-specific double-stranded cDNAs can then be inserted into a plasmid or viral vector, which is then introduced into a host bacterial, yeast, animal or plant cell. The host cells are then grown in culture media, resulting in a population of host cells containing (or in many cases, expressing) the gene of interest.
This entire process, from isolation of mRNA to insertion of the cDNA into a plasmid or vector to growth of host cell populations containing the isolated gene, is termed xe2x80x9ccDNA cloning.xe2x80x9d If cDNAs are prepared from a number of different mRNAs, the resulting set of cDNAs is called a xe2x80x9ccDNA library,xe2x80x9d an appropriate term since the set of cDNAs represents a xe2x80x9cpopulationxe2x80x9d of genes comprising the functional genetic information present in the source cell, tissue or organism. Genotypic analysis of these cDNA libraries can yield much information on the structure and function of the organisms from which they were derived.
Retroviral Reverse Transcriptase Enzymes
Three prototypical forms of retroviral RT have been studied thoroughly. Moloney Murine Leukemia Virus (M-MLV) RT contains a single subunit of 78 kDa with RNA-dependent DNA polymerase and RNase H activity. This enzyme has been cloned and expressed in a fully active form in E. coli (reviewed in Prasad, V. R., Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, p. 135 (1993)). Human Immunodeficiency Virus (HIV) RT is a heterodimer of p66 and p51 subunits in which the smaller subunit is derived from the larger by proteolytic cleavage. The p66 subunit has both a RNA-dependent DNA polymerase and an RNase H domain, while the p51 subunit has only a DNA polymerase domain. Active HIV p66/p51 RT has been cloned and expressed successfully in a number of expression hosts, including E. coli (reviewed in Le Grice, S. F. J., Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory press, p. 163 (1993)). Within the HIV p66/p51 heterodimer, the 51-kD subunit is catalytically inactive, and the 66-kD subunit has both DNA polymerase and RNase H activity (Le Grice, S. F. J., et al., EMBO Journal 10:3905 (1991), Hostomsky, Z., et al., J. Virol. 66:3179 (1992)). Avian Sarcoma-Leukosis Virus (ASLV) RT, which includes but is not limited to Rous Sarcoma Virus (RSV) RT, Avian Myeloblastosis Virus (AMV) RT, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV RT, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV RT, Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A RT, Avian Sarcoma Virus UR2 Helper Virus UR2AV RT, Avian Sarcoma Virus Y73 Helper Virus YAV RT, Rous Associated Virus (RAV) RT, and Myeloblastosis Associated Virus (MAV) RT, is also a heterodimer of two subunits, xcex1 (approximately 62 kDa) and xcex2 (approximately 94 kDa), in which a is derived from xcex2 by proteolytic cleavage (reviewed in Prasad, V. R., Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1993), p. 135). ASLV RT can exist in two additional catalytically active structural forms, xcex2xcex2 and xcex1 (Hizi, A. and Joklik, W. K., J. Biol. Chem. 252: 2281 (1977)). Sedimentation analysis suggests xcex1xcex20 and xcex2xcex2 are dimers and that the xcex1 form exists in an equilibrium between monomeric and dimeric forms (Grandgenett, D. P., et al., Proc. Nat. Acad. Sci. USA 70: 230 (1973); Hizi, A. and Joklik, W. K., J. Biol. Chem. 252: 2281 (1977); and Soltis, D. A. and Skalka, A. M., Proc. Nat. Acad. Sci. USA 85: 3372 (1988)). The ASLV xcex1xcex2 and xcex2xcex2 RTs are the only known examples of retroviral RT that include three different activities in the same protein complex: DNA polymerase, RNase H, and DNA endonuclease (integrase) activities (reviewed in Skalka, A. M., Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1993), p. 193). The xcex1 form lacks the integrase domain and activity.
Various forms of the individual subunits of ASLV RT have been cloned and expressed. These include a 98-kDa precursor polypeptide that is normally processed proteolytically to xcex2 and a 4-kDa polypeptide removed from the xcex2 carboxy end (Alexander, F., et al., J. Virol. 61: 534 (1987) and Anderson, D. et al., Focus 17:53 (1995)), and the mature xcex2 subunit (Weis, J. H. and Salstrom, J. S., U.S. Pat. No. 4,663,290 (1987); and Soltis, D. A. and Skalka, A. M., Proc. Nat. Acad. Sci. USA 85:3372 (1988)). Heterodimeric RSV xcex1xcex2 RT has also been purified from E. coli cells expressing a cloned RSV xcex2 gene (Chernov, A. P., et al., Biomed Sci. 2:49 (1991)). However, there have been no reports heretofore of the simultaneous expression of cloned ASLV RT xcex1 and xcex2 genes resulting in the formation of heterodimeric xcex1xcex2 RT.
Reverse Transcription Efficiency
As noted above, the conversion of mRNA into cDNA by RT-mediated reverse transcription is an essential step in the study of proteins expressed from cloned genes. However, the use of unmodified RT to catalyze reverse transcription is inefficient for at least two reasons. First, RT sometimes destroys an RNA template before reverse transcription is initiated, primarily due to the activity of intrinsic RNase H activity present in RT. Second, RT often fails to complete reverse transcription after the process has been initiated (Berger, S. L., et al., Biochemistry 22:2365-2372 (1983); Krug, M. S., and Berger, S. L., Meth. Enzymol. 152:316 (1987)). Removal of the RNase H activity of RT can eliminate the first problem and improve the efficiency of reverse transcription (Gerard, G. F., et al., FOCUS 11(4):60 (1989); Gerard, G. F., et al., FOCUS 14(3):91 (1992)). However RTs, including those forms lacking RNase H activity (xe2x80x9cRNase Hxe2x88x92xe2x80x9d forms), still tend to terminate DNA synthesis prematurely at certain secondary structural (Gerard, G. F., et al., FOCUS 11(4):60 (1989); Myers, T. W., and Gelfand, D. H., Biochemistry 30:7661 (1991)) and sequence (Messer, L. I., et al., Virol 146:146 (1985)); Abbotts, J., et al., J. Biol. Chem. 268:10312-10323 (1993)) barriers in nucleic acid templates.
Even in the most efficient reverse transcription systems available today, which use RNase Hxe2x88x92M-MLV RT, yields of total cDNA product generally do not exceed 50% of input mRNA and the fraction of the product that is full-length does not exceed 50%. The secondary structural and sequence barriers in the mRNA template, which as described above can give rise to these limitations, occur frequently at homopolymer stretches (Messer, L. I., et al., Virol. 146:146 (1985); Huber, H. E., et al., J. Biol. Chem. 264:4669-4678 (1989); Myers, T. W., and Gelfand, D. H., Biochemistry 30:7661 (1991)), are more often sequence rather than secondary structural barriers (Abbotts, J., et al., J. Biol. Chem. 268:10312-10323 (1993)), and are often distinct for different RTs (Abbotts, J., et al., J. Biol. Chem. 268:10312-10323 (1993)). If these barriers could be overcome, yield of total and full-length cDNA product in reverse transcription reactions could be increased.
The present invention provides reverse transcriptase enzymes, compositions comprising such enzymes and methods useful in overcoming the above-described cDNA length limitations. In general, the invention provides compositions for use in reverse transcription of a nucleic acid molecule comprising two or more different polypeptides having reverse transcriptase activity. Such compositions may further comprise one or more nucleotides, a suitable buffer, and/or one or more DNA polymerases. The compositions of the invention may also comprise one or more oligonucleotide primers. Each reverse transcriptase used in the compositions of the invention may have a different transcription pause site on a given mRNA molecule. The reverse transcriptases in these compositions preferably are reduced or substantially reduced in RNase H activity, and most preferably are enzymes selected from the group consisting of Moloney Murine Leukemia Virus (M-MLV) Hxe2x88x92 reverse transcriptase, Rous Sarcoma Virus (RSV) Hxe2x88x92 reverse transcriptase, Avian Myeloblastosis Virus (AMV) Hxe2x88x92 reverse transcriptase, Rous Associated Virus (RAV) Hxe2x88x92 reverse transcriptase, Myeloblastosis Associated Virus (MAV) Hxe2x88x92 reverse transcriptase and Human Immunodeficiency Virus (HIV) Hxe2x88x92 reverse transcriptase or other ASLV Hxe2x88x92 reverse transcriptases. In preferred compositions, the reverse transcriptases are present at working concentrations.
The invention is also directed to methods for making one or more nucleic acid molecules, comprising mixing one or more nucleic acid templates (preferably one or more RNA templates and most preferably one or more messenger RNA templates) with two or more polypeptides having reverse transcriptase activity and incubating the mixture under conditions sufficient to make a first nucleic acid molecule or molecules complementary to all or a portion of the one or more nucleic acid templates. In a preferred embodiment, the first nucleic acid molecule is a single-stranded cDNA. Nucleic acid templates suitable for reverse transcription according to this aspect of the invention include any nucleic acid molecule or population of nucleic acid molecules (preferably RNA and most preferably mRNA), particularly those derived from a cell or tissue. In a preferred aspect, a population of mRNA molecules (a number of different mRNA molecules, typically obtained from cells or tissue) are used to make a cDNA library, in accordance with the invention. Preferred cellular sources of nucleic acid templates include bacterial cells, fungal cells, plant cells and animal cells.
The invention also concerns methods for making one or more double-stranded nucleic acid molecules. Such methods comprise (a) mixing one or more nucleic acid templates (preferably RNA or mRNA, and more preferably a population of mRNA templates) with two or more polypeptides having reverse transcriptase activity; (b) incubating the mixture under conditions sufficient to make a first nucleic acid molecule or molecules complementary to all or a portion of the one or more templates; and (c) incubating the first nucleic acid molecule under conditions sufficient to make a second nucleic acid molecule or molecules complementary to all or a portion of the first nucleic acid molecule or molecules, thereby forming one or more double-stranded nucleic acid molecules comprising the first and second nucleic acid molecules. Such methods may include the use of one or more DNA polymerases as part of the process of making the one or more double-stranded nucleic acid molecules. The invention also concerns compositions useful for making such double-stranded nucleic acid molecules. Such compositions comprise two or more reverse transcriptases and optionally one or more DNA polymerases, a suitable buffer and one or more nucleotides.
The invention also relates to methods for amplifying a nucleic acid molecule. Such amplification methods comprise mixing the double-stranded nucleic acid molecule or molecules produced as described above with one or more DNA polymerases and incubating the mixture under conditions sufficient to amplify the double-stranded nucleic acid molecule. In a first preferred embodiment, the invention concerns a method for amplifying a nucleic acid molecule, the method comprising (a) mixing one or more nucleic acid templates (preferably one or more RNA or mRNA templates and more preferably a population of mRNA templates) with two or more different polypeptides having reverse transcriptase activity and with one or more DNA polymerases and (b) incubating the mixture under conditions sufficient to amplify nucleic acid molecules complementary to all or a portion of the one or more templates. Preferably, the reverse transcriptases are reduced or substantially reduced in RNase H activity and the DNA polymerases comprise a first DNA polymerase having 3xe2x80x2 exonuclease activity and a second DNA polymerase having substantially reduced 3xe2x80x2 exonuclease activity. The invention also concerns compositions comprising two or more reverse transcriptases and one or more DNA polymerases for use in amplification reactions. Such compositions may further comprise one or more nucleotides and a buffer suitable for amplification. The compositions of the invention may also comprise one or more oligonucleotide primers.
In accordance with the invention, at least two, at least three, at least four, at least five, at least six, or more, reverse transcriptases may be used. Preferably, two to six, two to five, two to four, two to three, and most preferably two, reverse transcriptases are used in the compositions and methods of the invention. Such multiple reverse transcriptases may be added simultaneously or sequentially in any order to the compositions or in the methods of the invention. Alternatively, multiple different reactions with different enzymes may be performed separately and the reaction products may be mixed. Thus, the invention relates to the synthesis of the nucleic acid molecules by the methods of the invention in which multiple reverse transcriptases are used simultaneously or sequentially or separately. In particular, the invention relates to a method of making one or more nucleic acid molecules comprising incubating one or more nucleic acid templates (preferably one or more RNA templates or mRNA templates, and more preferably a population of mRNA templates) with a first reverse transcriptase under conditions sufficient to make one or more nucleic acid molecules complementary to all or a portion of the one or more templates. In accordance with the invention, the one or more nucleic acid molecules (including mRNA templates and/or synthesized nucleic acid molecules) may be incubated with a second reverse transcriptase under conditions sufficient to make additional nucleic acid molecules complementary to all or a portion of the templates or to increase the length of the previously made nucleic acid molecules. In accordance with the invention, this procedure may be repeated any number of times with the same or different reverse transcriptases of the invention. For example, the first and second reverse transcriptases may be the same or different. Furthermore, the first and third reverse transcriptases (in aspects of the invention where the procedure is repeated three times using a first, second, and third reverse transcriptase) may be the same while the second reverse transcriptase may be different from the first and the third reverse transcriptase. Thus, any combination of the same and/or different reverse transcriptases may be used in this aspect of the invention. Preferably, when multiple reverse transcriptases are used, at least two reverse transcriptases are different.
In a related aspect of the invention, the reverse transcriptase used in the reaction may retain all or a portion of its activity during subsequent reaction steps. Alternatively, the reverse transcriptase used in the reaction may be inactivated by any method prior to incubation with additional reverse transcriptases. Such an inactivation may include but is not limited to heat inactivation, organic extraction (e.g., with phenol and/or chloroform), ethanol precipitation and the like.
The synthesized nucleic acid molecules made by simultaneous or sequential or separate addition of reverse transcriptases may then be used to make double stranded nucleic acid molecules. Such synthesized nucleic acid molecules serve as a template which when incubated under appropriate conditions (e.g., preferably in the presence of one or more DNA polymerases) make nucleic acid molecules complementary to all or a portion of the synthesized nucleic acid molecules, thereby forming a number of double stranded nucleic acid molecules. The double stranded molecules may then be amplified in accordance with the invention.
The invention is also directed to nucleic acid molecules (particularly single- or double-stranded cDNA molecules) or amplified nucleic acid molecules produced according to the above-described methods and to vectors (particularly expression vectors) comprising these nucleic acid molecules or amplified nucleic acid molecules.
The invention is also directed to recombinant host cells comprising the above-described nucleic acid molecules, amplified nucleic acid molecules or vectors. Preferred such host cells include bacterial cells, yeast cells, plant cells and animal cells (including insect cells and mammalian cells).
The invention is further directed to methods of producing a polypeptide comprising culturing the above-described recombinant host cells and isolating the polypeptide, and to a polypeptide produced by such methods.
The invention also concerns methods for sequencing one or more nucleic acid molecules using the compositions or enzymes of the invention. Such methods comprise (a) mixing one or more nucleic acid molecules (e.g., one or more RNA or DNA molecules) to be sequenced with one or more primers, one or more polypeptides having reverse transcriptase activity, one or more nucleotides and one or more terminating agents, such as one or more dideoxynucleoside triphosphates; (b) incubating the mixture under conditions sufficient to synthesize a population of nucleic acid molecules complementary to all or a portion of the one or more nucleic acid molecules to be sequenced; and (c) separating the population of nucleic acid molecules to determine the nucleotide sequence of all or a portion of the one or more nucleic acid molecules to be sequenced. In these sequencing methods of the invention, the one or more polypeptides having reverse transcriptase activity may be added simultaneously, sequentially, or separately to the reaction mixtures as described above.
The invention is also directed to kits for use in the methods of the invention. Such kits can be used for making, sequencing or amplifying nucleic acid molecules (single- or double-stranded). The kits of the invention comprise a carrier, such as a box or carton, having in close confinement therein one or more containers, such as vials, tubes, bottles and the like. In the kits of the invention, a first container contains one or more of the reverse transcriptase enzymes (preferably one or more such enzymes that are reduced or substantially reduced in RNase H activity) or one or more of the compositions of the invention. In another aspect, the kit may contain one or more containers comprising two or more, three or more, four or more, five or more, six or more, and the like, reverse transcriptases, preferably one or more containers comprising two to six, two to five, two to four, two to three, or more preferably two, reverse transcriptases. The kits of the invention may also comprise, in the same or different containers, one or more DNA polymerase (preferably thermostable DNA polymerases), a suitable buffer for nucleic acid synthesis and one or more nucleotides. Alternatively, the components of the composition may be divided into separate containers (e.g., one container for each enzyme). In preferred kits of the invention, the reverse transcriptases are reduced or substantially reduced in RNase H activity, and are most preferably selected from the group consisting of M-MLV Hxe2x88x92 reverse transcriptase, RSV Hxe2x88x92 reverse transcriptase, AMV Hxe2x88x92 reverse transcriptase, RAV Hxe2x88x92 reverse transcriptase, MAV Hxe2x88x92 reverse transcriptase and HIV Hxe2x88x92 reverse transcriptase. In additional preferred kits of the invention, the enzymes (reverse transcriptases and/or DNA polymerases) in the containers are present at working concentrations.
The invention also relates to methods of producing RSV reverse transcriptase (and/or subunits thereof). In particular, the invention relates to methods for producing RSV reverse transcriptase (and/or subunits thereof) containing RNase H activity, to methods for producing RSV reverse transcriptase (and/or subunits thereof) that is reduced or substantially reduced in RNase H activity, and to RSV reverse transcriptases (and/or subunits thereof) produced by such methods.
The invention further relates to methods for using such reverse transcriptases and to kits comprising such reverse transcriptases. In particular, the RSV reverse transcriptases (and/or subunits thereof) of the invention may be used in methods of sequencing, amplification and production (via, e.g., reverse transcription) of nucleic acid molecules.
The invention also relates to methods of producing AMV reverse transcriptase (and/or subunits thereof). In particular, the invention relates to methods for producing AMV reverse transcriptase (and/or subunits thereof) containing RNase H activity, to methods for producing AMV reverse transcriptase (and/or subunits thereof) that is reduced or substantially reduced in RNase H activity, and to AMV reverse transcriptases (and/or subunits thereof) produced by such methods.
The invention further relates to methods for using such reverse transcriptases and to kits comprising such reverse transcriptases. In particular, the AMV reverse transcriptases (and/or subunits thereof of the invention may be used in methods of sequencing, amplification and production (via, e.g., reverse transcription) of nucleic acid molecules.
The invention also generally relates to methods of producing ASLV reverse transcriptases (and/or subunits thereof). In particular, the invention relates to methods for producing ASLV reverse transcriptases (and/or subunits thereof) containing RNase H activity, to methods for producing such ASLV reverse transcriptases that are reduced or substantially reduced in RNase H activity, and to ASLV reverse transcriptases produced by such methods.
The invention further relates to methods for using such reverse transcriptases and to kits comprising such reverse transcriptases. In particular, the ASLV reverse transcriptases (and/or subunits thereof) of the invention may be used in methods of sequencing, amplification and production (e.g., via reverse transcription) of nucleic acid molecules.
The invention further relates to methods for elevated- or high-temperature reverse transcription of a nucleic acid molecule comprising (a) mixing one or more nucleic acid templates (preferably one or more RNA molecules (e.g., one or more mRNA molecules or polyA+RNA molecules, and more preferably a population of mRNA molecules) or one or more DNA molecules) with one or more polypeptides having reverse transcriptase activity; and (b) incubating the mixture at a temperature of 50xc2x0 C. or greater and under conditions sufficient to make a first nucleic acid molecule or molecules (such as a full length cDNA molecule) complementary to all or a portion of the one or more nucleic acid templates. In a preferred aspect, a population of mRNA molecules is used to make a cDNA library at elevated or high temperatures. In another aspect, elevated- or high-temperature nucleic acid synthesis is conducted with multiple reverse transcriptases (i.e., two or more, three or more, four or more, five or more, six or more, and the like, more preferably two to six, two to five, two to four, two to three, and still more preferably two, reverse transcriptases), which may be added to the reaction mixture simultaneously or sequentially or separately as described above. In preferred such methods, the mixture is incubated at a temperature of about 51xc2x0 C. or greater, about 52xc2x0 C. or greater, about 53xc2x0 C. or greater, about 54xc2x0 C. or greater, about 55xc2x0 C. or greater, about 56xc2x0 C. or greater, about 57xc2x0 C. or greater, about 58xc2x0 C. or greater, about 59xc2x0 C. or greater, about 60xc2x0 C. or greater, about 61xc2x0 C. or greater, about 62xc2x0 C. or greater, about 63xc2x0 C. or greater, about 64xc2x0 C. or greater, about 65xc2x0 C. or greater, about 66xc2x0 C. or greater, about 67xc2x0 C. or greater, about 68xc2x0 C. or greater, about 69xc2x0 C. or greater, about 70xc2x0 C. or greater, about 71xc2x0 C. or greater, about 72xc2x0 C. or greater, about 73xc2x0 C. or greater, about 74xc2x0 C. or greater, about 75xc2x0 C. or greater, about 76xc2x0 C. or greater, about 77xc2x0 C. or greater, or about 78xc2x0 C. or greater; or at a temperature range of from about 50xc2x0 C. to about 75xc2x0 C., about 51xc2x0 C. to about 75xc2x0 C., about 52xc2x0 C. to about 75xc2x0 C., about 53xc2x0 C. to about 75xc2x0 C., about 54xc2x0 C. to about 75xc2x0 C., about 55xc2x0 C. to about 75xc2x0 C., about 50xc2x0 C. to about 70xc2x0 C., about 51xc2x0 C. to about 70xc2x0 C., about 52xc2x0 C. to about 70xc2x0 C., about 53xc2x0 C. to about 70xc2x0 C., about 54xc2x0 C. to about 70xc2x0 C., about 55xc2x0 C. to about 70xc2x0 C., about 55xc2x0 C. to about 65xc2x0 C., about 56xc2x0 C. to about 65xc2x0 C., about 56xc2x0 C. to about 64xc2x0 C. or about 56xc2x0 C. to about 62xc2x0 C. The invention is also directed to such methods which further comprise incubating the first nucleic acid molecule or molecules under conditions sufficient to make a second nucleic acid molecule or molecules complementary to all or portion of the first nucleic acid molecule or molecules. According to the invention, the first and second nucleic acid molecules produced by these methods may be DNA molecules, and may form a double stranded DNA molecule or molecules which may be a full length cDNA molecule or molecules, such as a cDNA library. The one or more polypeptides having reverse transcriptase activity that are used in these methods preferably are reduced or substantially reduced in RNase H activity, and are preferably selected from ASLV reverse transcriptases (and/or subunits thereof) such as one or more subunits of AMV reverse transcriptase and/or one or more subunits of RSV reverse transcriptase and/or one or more subunits of MAV reverse transcriptase, and/or one or more subunits of RAV reverse transcriptase, particularly wherein the subunits are reduced or substantially reduced in RNase H activity.
The invention also relates to kits for elevated- or high-temperature nucleic acid synthesis, which may comprise one or more components selected from the group consisting of one or more reverse transcriptases (preferably one or more ASLV reverse transcriptases such as AMV or RSV reverse transcriptases (or one or more subunits thereof), and more preferably one or more AMV or RSV reverse transcriptases (or one or more subunits thereof) which are reduced or substantially reduced in RNase H activity), one or more nucleotides, one or more primers and one or more suitable buffers.
The invention is also directed to nucleic acid molecules produced by the above-described methods which may be full-length cDNA molecules, to vectors (particularly expression vectors) comprising these nucleic acid molecules and to host cells comprising these vectors and nucleic acid molecules.
Other preferred embodiments of the present invention will be apparent to one of ordinary skill in light of the following drawings and description of the invention, and of the claims.